Assay Overview: Pseudomonas Aeruginosa growth delay assay (absorbance) in presence of metal chelatorBacteria typically require total iron concentrations in the micromolar range to support growth. Many pathogens such as P. aeruginosa produce siderophores (e.g. pyoverdine) with molecular weights below 1500 Da that bind to iron with remarkably high affinities. The PvdQ acylase is responsible for removal of the fatty acid on pyoverdine. Interestingly, mutant strains of P. aeruginosa with a deletion in the PvdQ gene do not make pyoverdine and cannot grow on iron limiting media. This secondary assay aims to selectively identify small molecules delaying the growth of Pseudomonas Aeruginosa in presence of iron chelator ethylenediamine-di (o-hydroxyphenulacetic acid) EDDHA . The small molecules will reduce the absorbance signal observed at a wavelength of 600nm mediated by the growth or absorbance signal observed at a wavelength of 405nm (pyoverdine production) of Pseudomonas Aeruginosa in presence of EDDHA.

Expected outcome: Identification of probe(s) delaying growth of Pseudomonas Aeruginosa in presence of iron chelator EDDHA. Compounds reducing the absobance by 35% at 405nm and 600nm in duplicate will be considered hits.

ACTIVE CONCENTRATION LIMIT:For each sample, the highest valid tested concentration (Max_Concentration) was determined and the active concentration limit (AC_limit) was set to equal (2)(Max_Concentration).

NORMALIZATION:The raw signals of the plate wells were normalized using the 'Neutral Controls' method in Genedata Assay Analyzer (v7.0.3):The median raw signal of the intraplate neutral controls (NC) is set to a normalized activity value of 0.A normalized activity value of 100 is defined as (2)(NC).A normalized activity value of -50 is defined as (0.5)(NC).Experimental wells values were scaled to this range.

MEASUREMENT USED TO DETERMINE ACTIVE CONCENTRATION (AC): absACnn, the concentration at which the curve crosses threshold -35.0AC values were calculated using the curve fitting strategies in Genedata Screener Condoseo (7.0.3).AC values were calculated up to the active concentration limit described for each sample.pAC was set to equal -1*log10(AC)

PUBCHEM_ACTIVITY_SCORE:If PUBCHEM_ACTIVITY_OUTCOME = 1 (inactive) or 3 (inconclusive),then PUBCHEM_ACTIVITY_SCORE = 0If PUBCHEM_ACTIVITY_OUTCOME = 2 (active)then PUBCHEM_ACTIVITY_SCORE = (10)(pAC)Scores relate to AC in this manner:120 = 1 pM90 = 1 nM60 = 1 uM30 = 1 mM0 = 1 MWhen the active concentration (AC) is calculated to be greater than the highest valid tested concentration (Max_Concentration), the PUBCHEM_ACTIVITY_SCORE is calculated using Max_Concentration as the basis.When the active concentration (AC) is calculated to be less than the lowest tested concentration, the PUBCHEM_ACTIVITY_SCORE is calculated using the lowest tested concentration as the basis.

Note:The individual dose data point columns ('Activity_at_xxuM') reported here represent the median of valid (unmasked) replicate observations at each concentration. These values are the inputs to a curve fitting algorithm.All other data columns represent values which are derived during the curve fitting algorithm; this may sometimes include automatic further masking of some replicate data points.Occasionally this results in perceived inconsistencies: for example, between the derived 'Maximal_Activity' and the apparent most active data point.